RESUMO
Histone acetyltransferase p300/CBP-associated factor (PCAF) belonging to GCN5 family regulates various epigenetic events for transcriptional regulation through alterations in the chromatin structure. During normal development of B cells, gene expressions of numerous transcription factors are strictly regulated by epigenetic mechanisms including histone acetylation and deacetylation to complete their development pathways. Here, by analyzing PCAF-deficient DT40 mutants, ΔPCAF, we report that PCAF takes part in transcriptional activation of B cell lymphoma-6 (Bcl-6) and Paired box gene 5 (Pax5), which are essential transcription factors for normal development of B cells. PCAF-deficiency caused drastic decrease in mRNA levels of Bcl-6 and Pax5, and remarkable increase in that of B lymphocyte-induced maturation protein-1 (Blimp-1). In addition, chromatin immunoprecipitation assay showed that PCAF-deficiency caused remarkable decrease in acetylation levels of both H3K9 and H3K14 residues within chromatin surrounding the 5'-flanking regions of Bcl-6 and Pax5 genes in vivo, suggesting that their gene expressions may be regulated by PCAF. These results revealed that PCAF is involved in transactivation of Bcl-6 and Pax5 genes, resulting in down-regulation of Blimp-1 gene expression, and plays a key role in epigenetic regulation of B cell development.
Assuntos
Linfócitos B/metabolismo , Fator de Transcrição PAX5/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Ativação Transcricional , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Linhagem Celular , GalinhasRESUMO
The BCNT (Bucentaur) superfamily is classified by an uncharacteristic conserved sequence of â¼80 amino acids (aa) at the C-terminus, BCNT-C (the conserved C-terminal region of Bcnt/Cfdp1). Whereas the yeast Swc5 and Drosophila Yeti homologues play crucial roles in chromatin remodelling organization, mammalian Bcnt/Cfdp1 (craniofacial developmental protein 1) remains poorly understood. The protein, which lacks cysteine, is largely disordered and comprises an acidic N-terminal region, a lysine/glutamic acid/proline-rich 40 aa sequence and BCNT-C. It shows complex mobility on SDS/PAGE at â¼50 kDa, whereas its calculated molecular mass is â¼33 kDa. To characterize this mobility discrepancy and the effects of post-translational modifications (PTMs), we expressed various deleted His-Bcnt in E. coli and HEK cells and found that an acidic stretch in the N-terminal region is a main cause of the gel shift. Exogenous BCNT/CFDP1 constitutively expressed in HEK clones appears as a doublet at 49 and 47 kDa, slower than the protein expressed in Escherichia coli but faster than the endogenous protein on SDS/PAGE. Among seven in vivo phosphorylation sites, Ser(250), which resides in a region between disordered and ordered regions in BCNT-C, is heavily phosphorylated and detected predominantly in the 49 kDa band. Together with experiments involving treatment with phosphatases and Ser(250) substitutions, the results indicate that the complex behaviour of Bcnt/Cfdp1 on SDS/PAGE is caused mainly by an acidic stretch in the N-terminal region and Ser(250) phosphorylation in BCNT-C. Furthermore, Bcnt/Cfdp1 is acetylated in vitro by CREB-binding protein (CBP) and four lysine residues including Lys(268) in BCNT-C are also acetylated in vivo, revealing a protein regulated at multiple levels.
Assuntos
Fosfoproteínas/química , Fosfoproteínas/metabolismo , Acetilação , Substituição de Aminoácidos , Proteína de Ligação a CREB/química , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Epigênese Genética , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Proteínas Nucleares , Fosfoproteínas/genética , Fosforilação/fisiologia , Estrutura Terciária de ProteínaRESUMO
The transcription factor paired box gene 5 (Pax5) is essential for B cell development. In this study, complementation analyses in Pax5-deficient DT40 cells showed that three Pax5 isoforms Pax5A, Pax5B and Pax5BΔEx8 (another spliced isoform of Pax5B lacking exon 8) exhibit distinct roles in transcriptional regulation of six B cell development-related genes (activation-induced cytidine deaminase, Aiolos, BTB and CNC homology 2, B cell lymphoma-6, early B cell factor 1, origin binding factor-1 genes), transcriptions of which are remarkably down-regulated by Pax5-deficiency. Moreover, ectopic expression study shows that these Pax5 isoforms may regulate themselves and each other at the transcriptional level.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Fator de Transcrição PAX5/metabolismo , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/metabolismo , Animais , Linhagem Celular Transformada , Galinhas , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Fator de Transcrição PAX5/genética , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Transcrição GênicaRESUMO
The endoplasmic reticulum (ER), a complex membrane structure, has important roles in all eukaryotic cells. Catastrophe of its functions would lead to ER stress that causes various diseases such as cancer, neurodegenerative diseases, diabetes and so on. Prolonged ER stress could trigger apoptosis via activation of various signal transduction pathways. To investigate physiological roles of histone acetyltransferase GCN5 in regulation of ER stress, we analyzed responses of homozygous GCN5-deficient DT40 mutants, ΔGCN5, against ER stress. GCN5-deficiency in DT40 caused drastic resistance against apoptosis induced by pharmacological ER stress agents (thapsigargin and tunicamycin). Pharmaceutical analysis using specific Bcl-2 inhibitors showed that the drastic resistance against prolonged ER stress-induced apoptosis is, in part, due to up-regulation of Bcl-2 gene expression in ΔGCN5. These data revealed that GCN5 is involved in regulation of prolonged ER stress-induced apoptosis through controlling Bcl-2 gene expression.
Assuntos
Apoptose , Retículo Endoplasmático/metabolismo , Genes bcl-2 , Histona Acetiltransferases/metabolismo , Regulação para Cima , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Galinhas , Retículo Endoplasmático/efeitos dos fármacos , Histona Acetiltransferases/genética , Tapsigargina/farmacologiaRESUMO
The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD) indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy.
RESUMO
The histone acetyltransferase p300/CBP-associated factor (PCAF) catalyzes acetylation of core histones and plays important roles in epigenetics by altering the chromatin structure in vertebrates. In this study, PCAF-deficient DT40 mutants were analyzed and it was found that PCAF participates in regulation of secretory IgM heavy chain (H-chain) synthesis. Remarkably, PCAF-deficiency causes an increase in the amount of secretory IgM H-chain mRNA, but not in that of IgM light chain and membrane-bound IgM H-chain mRNAs, resulting in dramatic up-regulation of the amount of secretory IgM protein. These findings suggest that PCAF regulates soluble antibody production and is thus an effective suppressor of secretory IgM H-chain synthesis.
Assuntos
Regulação para Baixo , Imunoglobulina M/biossíntese , Células Precursoras de Linfócitos B/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Animais , Linhagem Celular , Galinhas , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/genética , Células Precursoras de Linfócitos B/enzimologia , Fatores de Transcrição de p300-CBP/genéticaRESUMO
Byproducts of cytokine activation are sometimes useful as surrogate biomarkers for monitoring cytokine generation in patients. Transforming growth factor (TGF)-ß plays a pivotal role in pathogenesis of hepatic fibrosis. TGF-ß is produced as part of an inactive latent complex, in which the cytokine is trapped by its propeptide, the latency-associated protein (LAP). Therefore, to exert its biological activity, TGF-ß must be released from the latent complex. Several proteases activate latent TGF-ß by cutting LAP. We previously reported that Camostat Mesilate, a broad spectrum protease inhibitor, which is especially potent at inhibiting plasma kallikrein (PLK), prevented liver fibrosis in the porcine serum-induced liver fibrosis model in rats. We suggested that PLK may work as an activator of latent TGF-ß during the pathogenesis of liver diseases in the animal models. However, it remained to be elucidated whether this activation mechanism also functions in fibrotic liver in patients. Here, we report that PLK cleaves LAP between R(58) and L(59) residues. We have produced monoclonal antibodies against two degradation products of LAP (LAP-DP) by PLK, and we have used these specific antibodies to immunostain LAP-DP in liver tissues from both fibrotic animals and patients. The N-terminal side LAP-DP ending at R(58) (R(58) LAP-DP) was detected in liver tissues, while the C-terminal side LAP-DP beginning at L(59) (L(59) LAP-DP) was not detectable. The R(58) LAP-DP was seen mostly in α-smooth muscle actin-positive activated stellate cells. These data suggest for the first time that the occurrence of a PLK-dependent TGF-ß activation reaction in patients and indicates that the LAP-DP may be useful as a surrogate marker reflecting PLK-dependent TGF-ß activation in fibrotic liver both in animal models and in patients.
RESUMO
GCN5 is involved in the acetylation of core histones, which is an important epigenetic event for transcriptional regulation through alterations in the chromatin structure in eukaryotes. To investigate physiological roles of GCN5, we have systematically analyzed phenotypes of homozygous GCN5-deficient DT40 mutants. Here, we report participation of GCN5 in regulation of IgM heavy chain (H-chain) gene expression. GCN5-deficiency down-regulates gene expressions of IgM H-chain (as whole, membrane-bound and secreted forms of its mRNA) but not light chain (L-chain), causing decreases in membrane-bound and secreted forms of IgM proteins. Chromatin immnoprecipitation assay revealed that GCN5 binds to the chicken IgM H-chain gene around its constant region but not L-chain gene, and acetylate Lys-9 residues of histone H3 within chromatin surrounding the constant region. These results suggest that GCN5 takes part in transcriptional regulation of the IgM H-chain gene via histone acetylation resulting in formation of relaxed chromatin arrangement around its coding region and plays a key role in epigenetic regulation of B cell functions.
Assuntos
Proteínas Aviárias/genética , Regulação da Expressão Gênica , Histona Acetiltransferases/genética , Cadeias Pesadas de Imunoglobulinas/genética , Células Precursoras de Linfócitos B/metabolismo , Acetilação , Animais , Proteínas Aviárias/metabolismo , Linhagem Celular Tumoral , Galinhas , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Regulação para Baixo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Immunoblotting , Cadeias Pesadas de Imunoglobulinas/metabolismo , Imunoglobulina M/genética , Imunoglobulina M/metabolismo , Lisina/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Células Precursoras de Linfócitos B/patologia , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In this study, we revealed that GCN5 and early B cell factor 1 (EBF1) participate in regulation of protein kinase Cθ (PKCθ) gene expression in an opposite manner in immature B cells. GCN5-deficiency in DT40 caused drastic down-regulation of transcription of PKCθ. In contrast, EBF1-deficiency brought about remarkable up-regulation of that of PKCθ, and re-expression of EBF1 dramatically suppressed transcription of PKCθ. Chromatin immunoprecipitation assay revealed that GCN5 binds to the 5'-flanking region of the chicken PKCθ gene and acetylates histone H3, and EBF1 binds to the 5'-flanking region of the gene surrounding putative EBF1 binding motifs.
Assuntos
Proteínas Aviárias/genética , Linfócitos B/enzimologia , Histona Acetiltransferases/fisiologia , Proteína Quinase C/genética , Transativadores/fisiologia , Região 5'-Flanqueadora , Acetilação , Animais , Proteínas Aviárias/metabolismo , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Galinhas , Regulação para Baixo , Repressão Enzimática , Histonas/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BRCA2 localizes to centrosomes between G1 and prophase and is removed from the centrosomes during mitosis, but the underlying mechanism is not clear. Here we show that BRCA2 is cleaved into two fragments by membrane type-1 matrix metalloproteinase (MT1-MMP), and that knockdown of MT1-MMP prevents the removal of BRCA2 from centrosomes during metaphase. Mass spectrometry mapping revealed that the MT1-MMP cleavage site of human BRCA2 is between Asn-2135 and Leu-2136 ((2132)LSNN/LNVEGG(2141)), and the point mutation L2136D abrogated MT1-MMP cleavage. Our data demonstrate that MT1-MMP proteolysis of BRCA2 regulates the abundance of BRCA2 on centrosomes.
Assuntos
Proteína BRCA2/metabolismo , Centrossomo/metabolismo , Metaloproteinase 14 da Matriz/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteína BRCA2/química , Proteína BRCA2/genética , Sítios de Ligação/genética , Ciclo Celular , Divisão Celular , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Metaloproteinase 14 da Matriz/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
During B-cell differentiation, the gene expression of B-cell differentiation-related transcription factors must be strictly controlled by epigenetic mechanisms including histone acetylation and deacetylation, to complete the differentiation pathway. GCN5, one of the most important histone acetyltransferases, is involved in epigenetic events for transcriptional regulation through alterations in the chromatin structure. In this study, by analyzing the homozygous DT40 mutants GCN5(-/-), generated with gene targeting techniques, we found that GCN5 was necessary for transcriptional activation of IRF-4, an essential transcription factor for plasma cell differentiation. GCN5 deficiency caused drastic decreases in both the mRNA and the protein levels of Blimp-1 and IRF-4. The ectopic expression of Blimp-1 and IRF-4 suggests that IRF-4, but not Blimp-1, is the target gene of GCN5 in immature B cells. Moreover, a chromatin immunoprecipitation assay showed that GCN5 bound to the IRF-4 gene around its 5'-flanking region and acetylated H3K9 residues within chromatin surrounding the region in vivo, suggesting that gene expression of IRF-4 is certainly regulated by GCN5. These results reveal that GCN5 is essential for IRF-4 gene expression, followed by transcriptional activation of Blimp-1, and plays a key role in epigenetic regulation of B-cell differentiation.
Assuntos
Proteínas Aviárias/genética , Linfócitos B/citologia , Diferenciação Celular/genética , Regulação da Expressão Gênica/imunologia , Fatores Reguladores de Interferon/genética , Fatores de Transcrição/imunologia , Ativação Transcricional , Fatores de Transcrição de p300-CBP/genética , Animais , Proteínas Aviárias/biossíntese , Diferenciação Celular/imunologia , Galinhas , Imunoprecipitação da Cromatina , Epigênese Genética , Técnicas de Inativação de Genes , Fatores Reguladores de Interferon/biossíntese , Células Precursoras de Linfócitos B/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ativação Transcricional/genéticaRESUMO
The Fas antigen, also designated as APO-1 or CD95, is a member of the tumor necrosis factor receptor superfamily and can mediate apoptotic cell death in various cells. We report here that blood coagulation factor XIII (plasma transglutaminase, fibrin stabilizing factor) inhibits apoptosis induced by a cytotoxic anti-Fas monoclonal antibody in Jurkat cells. When cells were treated with the antibody in fetal calf serum-containing media, higher-molecular-weight (180K) polypeptides containing Fas molecule were detected by immunoblotting. Under conditions where the transglutaminase activity was eliminated or suppressed, the cross-link of Fas was not observed, and concurrently cell death was hastened. Moreover, an antibody against factor XIII strongly accelerated the Fas-mediated apoptosis. Furthermore, addition of partially purified factor XIII neutralized the apoptosis-promoting effect of anti-factor XIII antibody, indicating that this enzyme is involved in cross-link of Fas and down-regulates Fas-mediated apoptotic cell death. Significantly, the cross-link of Fas was seen only in fetal calf serum but not in newly-born calf serum, 1-year-old calf serum or adult bovine serum. These data suggest that plasma transglutaminase factor XIII may play a key role in fetal development of vertebrates via cross-link of Fas antigen.
Assuntos
Apoptose , Fator XIIIa/metabolismo , Feto/metabolismo , Receptor fas/metabolismo , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Murinos , Catálise , Regulação para Baixo , Desenvolvimento Fetal , Humanos , Células Jurkat , Receptor fas/antagonistas & inibidoresRESUMO
By UV-irradiation, cells are subjected to DNA damage followed by mutation, cell death and/or carcinogenesis. DNA repair systems such as nucleotide excision repair (NER) and translesion DNA synthesis (TLS) protect cells against UV-irradiation. To understand the role of histone acetyltransferase GCN5 in regulation of DNA repair, we studied the sensitivity of GCN5-deficient DT40, GCN5(-/-), to various DNA-damaging agents including UV-irradiation, and effects of GCN5-deficiency on the expression of NER- and TLS-related genes. After UV-irradiation, cell death and DNA fragmentation of GCN5(-/-) were appreciably accelerated as compared with those of DT40. Interestingly, GCN5(-/-) showed a remarkable sensitivity to only UV-irradiation but not to other DNA-damaging agents tested. Semiquantitative RT-PCR showed that transcription of DNA polymerase η (POLH) gene whose deficiency is responsible for a variant form of xeroderma pigmentosum was drastically down-regulated in GCN5(-/-) (to â¼25%). In addition, ectopic expression of human POLH in GCN5(-/-) dramatically reversed the sensitivity to UV-irradiation of GCN5(-/-) to almost the same level of wild type DT40. Moreover, chromatin immunoprecipitation assay revealed that GCN5 binds to the chicken POLH gene 5'-flanking region that contains a typical CpG island and acetylates Lys-9 of histone H3, but not Lys-14 in vivo. These data suggest that GCN5 takes part in transcription regulation of POLH gene through alterations in the chromatin structure by direct interaction with its 5'-flanking region, and protects vertebrate cells against UV-induced DNA damage via controlling POLH gene expression.
Assuntos
Fragmentação do DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação/efeitos da radiação , Animais , Morte Celular/genética , Morte Celular/efeitos da radiação , Linhagem Celular , Galinhas , Ilhas de CpG/genética , DNA/biossíntese , DNA/genética , Reparo do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica/genética , Histonas/genética , Histonas/metabolismo , Humanos , Ligação Proteica/genética , Ligação Proteica/efeitos da radiação , Transcrição Gênica/genética , Transcrição Gênica/efeitos da radiação , Fatores de Transcrição de p300-CBP/genéticaRESUMO
Tardigrades are able to tolerate almost complete dehydration by reversibly switching to an ametabolic state. This ability is called anhydrobiosis. In the anhydrobiotic state, tardigrades can withstand various extreme environments including space, but their molecular basis remains largely unknown. Late embryogenesis abundant (LEA) proteins are heat-soluble proteins and can prevent protein-aggregation in dehydrated conditions in other anhydrobiotic organisms, but their relevance to tardigrade anhydrobiosis is not clarified. In this study, we focused on the heat-soluble property characteristic of LEA proteins and conducted heat-soluble proteomics using an anhydrobiotic tardigrade. Our heat-soluble proteomics identified five abundant heat-soluble proteins. All of them showed no sequence similarity with LEA proteins and formed two novel protein families with distinct subcellular localizations. We named them Cytoplasmic Abundant Heat Soluble (CAHS) and Secretory Abundant Heat Soluble (SAHS) protein families, according to their localization. Both protein families were conserved among tardigrades, but not found in other phyla. Although CAHS protein was intrinsically unstructured and SAHS protein was rich in ß-structure in the hydrated condition, proteins in both families changed their conformation to an α-helical structure in water-deficient conditions as LEA proteins do. Two conserved repeats of 19-mer motifs in CAHS proteins were capable to form amphiphilic stripes in α-helices, suggesting their roles as molecular shield in water-deficient condition, though charge distribution pattern in α-helices were different between CAHS and LEA proteins. Tardigrades might have evolved novel protein families with a heat-soluble property and this study revealed a novel repertoire of major heat-soluble proteins in these anhydrobiotic animals.
Assuntos
Desidratação/metabolismo , Proteínas/metabolismo , Tardígrados/metabolismo , Sequência de Aminoácidos , Animais , Estrutura Secundária de Proteína , ProteômicaRESUMO
Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.
Assuntos
Membrana Celular/metabolismo , Endossomos/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Nexinas de Classificação/metabolismo , Motivos de Aminoácidos , Animais , Transporte Biológico Ativo/fisiologia , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Endossomos/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Domínios PDZ , Ligação Proteica , RNA Interferente Pequeno , Nexinas de Classificação/genéticaRESUMO
The high-affinity choline transporter (CHT1), which is specifically expressed in cholinergic neurons, constitutes a rate-limiting step for acetylcholine synthesis. We have found that the exogenous ubiquitin ligase Nedd4-2 interacts with CHT1 expressed in HEK293 cells decreasing the amount of cell surface CHT1 by approximately 40%, and that small interfering RNA for endogenous Nedd4-2 enhances the choline uptake activity by CHT1 in HEK293 cells. These results indicate that Nedd4-2-mediated ubiquitination regulates the cell surface expression of CHT1 in cultured cells and suggest a possibility that treatments or drugs which inhibit the interaction between CHT1 and Nedd4-2 might be useful for diseases involving decrease in acetylcholine level such as Alzheimer's disease.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação da Expressão Gênica , Simportadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Acetilcolina/biossíntese , Biotinilação , Membrana Celular/genética , Membrana Celular/metabolismo , Neurônios Colinérgicos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HEK293 , Humanos , Imunoprecipitação/métodos , Ubiquitina-Proteína Ligases Nedd4 , Interferência de RNA , Análise de Sequência de DNA , Simportadores/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads-one recognizes N-acetyllactosamine (Galß1-4GlcNAc-), and the other recognizes the Lewis X structure (Galß1-4(Fucα1-3)GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane.
Assuntos
Proteínas ADAM/química , Amino Açúcares/química , Membrana Celular/química , Receptores de Superfície Celular/química , Espermatozoides/química , Sequência de Aminoácidos , Animais , Masculino , Dados de Sequência Molecular , Proteômica , SuínosRESUMO
Histone acetyltransferase(s) (HATs) are involved in the acetylation of core histones, which is an important event for transcription regulation through alterations in the chromatin structure in eukaryotes. General control non-depressible 5 (GCN5) was first identified as a global coactivator and transcription-related HAT. Here we report that GCN5 regulates the activation of phosphatidylinositol 3-kinase (PI3K)/acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) survival pathway in B cells exposed to oxidative stress via controlling gene expressions of spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk). The GCN5-deficiency remarkably caused apoptotic cell death by treatment with exogenous hydrogen peroxide (H(2)O(2)) in chicken DT40 cells. In GCN5-deficient DT40 cells, gene expressions of Syk and Btk, which are involved in activation of PI3K/Akt survival pathway in DT40 cells exposed to exogenous H(2)O(2), were remarkably decreased compared with those in wild type DT40 cells. In addition, phosphorylation of Akt in H(2)O(2)-treated GCN5-deficient cells was remarkably suppressed as compared to that of DT40. Chromatin immunoprecipitation assay revealed that GCN5 binds to proximal 5'-upstream regions of Syk and Btk genes in vivo. These results suggest that GCN5 takes part in transcriptional regulations of the Syk and Btk genes, and plays a key role in epigenetic regulation of PI3K/Akt survival pathway in B cells exposed to reactive oxygen species such as H(2)O(2).
Assuntos
Linfócitos B/fisiologia , Regulação Enzimológica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/biossíntese , Fatores de Transcrição de p300-CBP/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Apoptose , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Linhagem Celular , Galinhas , Imunoprecipitação da Cromatina , Ativação Enzimática , Peróxido de Hidrogênio/farmacologia , Mutação , Quinase Syk , Fatores de Transcrição de p300-CBP/genéticaRESUMO
The breast cancer susceptibility gene (BRCA2) is localized mainly in the nucleus where it plays an important role in DNA damage repair. Some BRCA2 protein is also present in the centrosome. Here, we demonstrate that BRCA2 interacts with plectin, a cytoskeletal cross-linker protein, and that this interaction controls the position of the centrosome. Phosphorylation of plectin by cyclin-dependent kinase 1/cyclin B (CDK1/CycB) kinase has been reported to abolish its cross-linking function during mitosis. Here, we induced phosphorylation of plectin in prepared fractions of HeLa cells by adding activated CDK1/CycB kinase. Consequently, there was significant dissociation of the centrosome from the nuclear membrane. Plectin has six homologous ankyrin-like repeat domains (termed PLEC M1-M6). Using a pull-down assay, we found that GST-PLEC M1 and a GST-C-terminal region fusion protein (which comprised PLEC M6, along with an adjacent vimentin site) interacted with BRCA2. Since each PLEC module exhibits high homology to the others, the possibility of all six domains participating in this interaction was indicated. Moreover, when PLEC M1 was overexpressed in HeLa cells, it competed with endogenous plectin and inhibited the BRCA2-plectin interaction. This inhibitory effect resulted in dissociation of the centrosomes from the nucleus and increased the rate of micronuclei formation which may lead to carcinogenesis. In addition, when either BRCA2 or plectin was suppressed by the appropriate siRNA, a similar change in centrosomal positioning was observed. We suggest that the BRCA2-plectin interaction plays an important role in the regulation of centrosome localization and also that displacement of the centrosome may result in genomic instability and cancer development.
Assuntos
Proteína BRCA2/fisiologia , Centrossomo/fisiologia , Plectina/fisiologia , Proteínas Reguladoras de Apoptose , Proteína Quinase CDC2/fisiologia , Linhagem Celular Tumoral , Núcleo Celular/patologia , Ciclina B/fisiologia , Ciclina B1 , Humanos , Imunoprecipitação , Neoplasias/etiologia , Plectina/química , Estrutura Terciária de ProteínaRESUMO
There is accumulating evidence that oxidative stress plays an important role in aging. Our previous phosphoproteomic study of the human neuroblastoma cell line SH-SY5Y revealed changes in the phosphorylation of several proteins such as lamin A/C during 6-hydroxydopamine-induced oxidative stress. The present study employed native proteomic analysis to clarify protein-protein interaction under physiological conditions. We examined oxidative stress-related changes in SH-SY5Y cellular proteins using blue-native polyacrylamide gel electrophoresis (BN-PAGE), a powerful tool for the separation of protein complexes. BN-PAGE gel images showed successful separation of several complexes. Components of these complexes, separated by 2-D BN-PAGE in combination with SDS-PAGE, were identified by peptide mass fingerprinting employing MALDI-TOF MS and an MS/MS ion search on LC-MS/MS. TCP-1 complex, ATP synthase, and the complex of heat shock protein 90 with its client proteins such as pyruvate kinase were detected. Two dimensional BN-PAGE and Western blot analysis revealed an increase of lamin A/C associated with heat shock protein 90 in response to 6-OHDA-induced oxidative stress.